Phytate Phosphorus Hydrolysis As Influenced by Dietary Calcium and

as inorganic (IMM) or an equivalent level as micro-mineral-amino acid complexes (MAAC). Adding. Ca or micro-minerals reduced (P < 0.05) PP hydrolysis ...
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J. Agric. Food Chem. 2003, 51, 4687−4693

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Phytate Phosphorus Hydrolysis As Influenced by Dietary Calcium and Micro-Mineral Source in Broiler Diets NADA M. TAMIM

AND

ROSELINA ANGEL*

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland, 20742

Phytate phosphorus (PP) hydrolysis by a 3-phytase was studied in vitro at pH 2.5 and 6.5 with either 0, 1.0, 4.0, or 9.0 g of Ca/kg diet, or 0, 1.0, 5.0, 7.5, or 10.0 g/kg diet of micro-mineral premix added as inorganic (IMM) or an equivalent level as micro-mineral-amino acid complexes (MAAC). Adding Ca or micro-minerals reduced (P < 0.05) PP hydrolysis at both pHs; however, the effect was greater at pH 6.5. An in vivo experiment was conducted in which broilers were fed one of six diets for 30 h. The experimental design was a factorial of three micro-mineral forms (0 added, IMM, and MAAC) and two Ca levels (0 or 5 g/kg). Adding Ca reduced (P < 0.05) PP disappearance and increased Ca apparent absorption. No micro-minerals effect (P > 0.05) was seen. Therefore, in poultry diets, it is Ca that inhibits PP hydrolysis and decreases P availability. KEYWORDS: Phytate phosphorus; calcium; absorption; micro-minerals; broilers

INTRODUCTION

Phytic acid (PA) (myo inositol hexaphosphoric acid) occurs naturally in plants, primarily in seeds, and serves as a storage form of phosphorus (P) (1). Poultry diets are composed mainly of plant based ingredients that have 60-90% of their P as phytate P (PP) (1, 2), which is poorly available (3, 4). This poor availability of the P present naturally in plant-based diets means that inorganic sources of P have to be added to the diet to meet the animal’s P requirement. The addition of inorganic P to diets that already may contain enough P, albeit in an unavailable form, results in diets that contain total P levels well in excess of requirement. This results in excess P, regardless of dietary source, being excreted. The presence of high levels of P in poultry excreta is currently a concern, especially when excreta is applied to soil as a fertilizer, because high excreta P can lead to soil P accumulation and increased P leaching or runoff into waterways. Poultry excreta management, primarily due to its proportionately high P content, has become a cost for poultry production and one of the factors for regulation of animal feeding operations. In its acidic form, PA has the capacity to bind or chelate multivalent cations including calcium (Ca), zinc (Zn), iron (Fe), magnesium (Mg), manganese (Mn), cobalt (Co), and copper (Cu) (1, 5, 6). Several researchers have observed that solubility of PA metal complexes is pH dependent (7, 8). Most phytatemineral complexes are soluble at low pHs (less than 3.5) with maximum insolubility occurring between pH 4 and 7 (7). Calcium and Mg phytate complexes precipitate at pHs between 4 and 6, and 5 and 7, respectively (8). The approximate pH of the intestine, where absorption of metal ions takes place, coincides with the pHs at which these complexes precipitate * To whom correspondence should be addressed. Telephone: (301) 4058494. Fax: (301) 314-9059. E-mail: [email protected].

(8). Precipitated phytate-mineral complexes are not accessible for hydrolysis or absorption in the intestine. The order of stability of metal-phytate complexes was found to be Cu > Zn > Co > Mn > Fe > Ca (2). Other researchers (6) reported the order of mineral potency as inhibitors of PP hydrolysis at neutral pH to be Zn2+ . Fe2+ > Mn2+ > Fe3+ > Ca2+ > Mg2+. Even though Ca has one of the lowest affinities for phytate, it has the greatest impact, because it is the mineral present at the highest concentration in the diet. Taylor (9) suggested that the primary factor determining PP utilization is the Ca ion concentration in the small intestine, where insoluble Ca-phytate complexes form. Increasing Ca level in the broiler diet from 1.2 to 5.2 g/kg decreased (P < 0.05) PP hydrolysis (10). Furthermore, high levels of Ca or Mg were found to lower the activities of intestinal alkaline phosphatase and phytase in broiler chicks, but Ca had a much more pronounced effect (11). Phytate P availability has been found to improve by the addition of some amino acids (1). The addition of certain amino acids, including cysteine and histidine, was found to alleviate some of the Zn deficiency syndrome signs (12). In general, amino acids, which possess one amino and one carboxyl group and can form neutral chelates, have the potential to be and fulfill the requirements of a chelating agent (13). Specific amino acid metal chelates have the metal embedded in a cyclic configuration bearing no electrochemical charges to bind any other compound, thus eliminating metal to phytate complexing potential (13). The improvement in mineral absorption seen by Nielsen (12) may be due to the amino acids having higher affinity for the minerals, therefore minimizing phytate mineral interactions. Fouad (13) reported that mineral amino acid chelates, if properly prepared, would be absorbed intact through the intestinal mucosa. This absorption of intact mineral amino acid complexes would be important to minimize any potential interaction between mineral ions and phytate if minerals are released while still in the digesta.

10.1021/jf034122x CCC: $25.00 © 2003 American Chemical Society Published on Web 07/03/2003

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The present study was designed to test the hypothesis that decreasing Ca in the diet as well as the use of micro-mineralamino acid complexes will increase PP hydrolysis both in vitro and in vivo. Experiments were conducted to determine the effect of Ca level and micro-mineral source (inorganic or amino acidmineral complexes) on PP hydrolysis in vitro at 2 different pHs and in vivo. MATERIALS AND METHODS Materials. Sodium acetate, glycine, calcium carbonate, sodium phytate (dodecasodium salt from rice), phytase enzyme (3-phytase from Aspergillus ficuum), and inorganic P were purchased from Sigma, St. Louis, MO. Inorganic micro-mineral premix (IMM) was purchased from Southern States Cooperative Inc., Richmond, VA, and was labeled to contain, per kg, 98 g of Ca from CaCO3, 210 g of Zn from ZnO, 120 g of Mn, of which 50% came from MnO and 50% from MnSO4, 40 g of Fe from FeSO4, 20 g of Cu from CuO, 3 g of I from Ca(IO3)2, and 0.05 g of Co from CoCO3. The IMM was analyzed by inductive coupled plasma (14) and was found to contain on an as-is basis: 116.4 g of Ca, 192.0 g of Zn, 107.5 g of Mn, 34.9 g of Fe and 17.3 g of Cu per kg of premix. Dry matter content was also determined to be 995 g/kg. Each of the following micromineral products was obtained as individual mineral products: Availa-Zn 100, zinc-amino acid complex; Availa-Mn 80, manganese-amino acid complex; Availa-Fe 60, ironamino acid complex; and Availa-Cu 100, copper-amino acid complex; from Zinpro Corporation, Eden Prairie, MN; CaI2O6 and CoCl2 from Sigma, St. Louis, MO. To make the micro-mineral-amino acid complex premix (MAAC), the amino acid complexes as well as CaI2O6 and CoCl2 were mixed such that the proportions of Zn, Mn, Fe, Cu, I, and Co were the same as those in the IMM. The MAAC was analyzed by inductive coupled plasma and found to contain, per kg, 60.6 g of Ca, 47.2 g of Zn, 24.9 g of Mn, 10.7 g of Fe and 5.3 g of Cu per kg of premix. Dry matter content was also determined to be 981 g/kg. By calculation, 4.5 g of MAAC would provide the same amount of micro-minerals (Fe, Mg, Cu, Zn, I, and Co) as 1 g of IMM. In Vitro Phytate Phosphorus Hydrolysis. For the in vitro work, the general procedures of Chen (15) were followed with some modifications, including incubation times (15, 30, 60, and 120 min, instead of 60 min only), sodium phytate concentration (4.62 g/L instead of 8.4 g/L), as well as the use of inorganic P as a standard, instead of P liberated by a phytase enzyme with known activity. All incubations were done in quadruplicate, and each one of these quadruplicate incubations served as the experimental unit. Phytate P hydrolysis by a 3-phytase enzyme (from Aspergillus ficuum) with a pH optimum of 5 (16) was determined over a 120 min period at pH 2.5, simulating gastric pH, and at pH 6.5, simulating small intestinal pH. Assuming typical PP content in a corn-soybean meal broiler starter diet of 2.7 g PP/kg and an expected feed to water consumption ratio of 2:1, a 4.62 g/L sodium phytate solution (929 mg PP/L) was prepared in a 200 mM glycine buffer (pH 2.5) or 200 mM sodium acetate buffer (pH 6.5), and this solution was used as the substrate. Buffers were chosen because of their pH specificities, with the glycine buffer being effective between pH 2.0 and 3.0 and the sodium acetate buffer between pH 3.5 and 6.5 (16). Phytase enzyme was suspended in a buffer and then diluted such that a 100-µL volume would contain the equivalent of 500 units (U) phytase/kg diet when added to the substrate solutions. The chosen level of phytase, 500 U phytase/kg diet, is within the range (300-800 U phytase/kg diet) recommended to be added to poultry diets (17, 18). A U of phytase activity, as defined by the manufacturer, is the amount of phytase needed to liberate 1.0 µmole of inorganic P from 4.2 × 10-2 M Mg ‚ K phytate per min at 37 °C and pH 2.5. Effect of Calcium. Four Ca levels were selected (0, 0.86, 3.44, and 7.74 g of CaCO3/L) to be equivalent to 0, 1.0, 4.0, or 9.0 g/kg diet. The highest level chosen, 9.0 g/kg, is the level most commonly used in broiler starter diets. The two other levels (4.0 and 1.0 g/kg) were chosen as intermediate and low levels. The four Ca levels were added to the Na-phytate substrate, and the pH of the solutions was checked

Tamim and Angel Table 1. Ingredient Composition and Nutrient Levels in the Basal Diet ingredient

basal g/kg

corn soybean meal, 48% crude soy oil vitamin mixa choline chloride, 60% salt DL methionine

541.8 398.7 51.1 0.8 0.8 4.6 2.2

formulated (analyzed) nutrient level crude protein (g/kg) 23.6 crude fat (g/kg) 7.02 energy (kcal ME/kg) 3230 calcium (g/kg) 1.8 (1.8) total phosphorus (g/kg) 4.1 (4.0) b non pp (g/kg) 1.3 (1.1) a Supplied the following per kilogram of feed: vitamin A, 14991 IU as retinyl acetate; vitamin D, 5291 ICU as cholecalciferol; vitamin E, 52.9 IU as DL-Rtocopheryl acetate; vitamin B12, 0.026 mg as cyanocobalamin; riboflavin, 17.64 mg as riboflavin; niacin, 70.55 mg as nicotinic acid; D-pantothenic acid, 24.6 mg as D-pantothenic acid; vitamin K, 3.2 mg as menadion sodium bisulfite complex; folic acid, 2.12 mg as folic acid; vitamin B6, 6.17 mg as pyridoxine hydrochloride; thiamine, 4.4 mg as thiamine mononitrate; and vitamin H, 0.149 mg as D-biotin. b Non phytate phosphorus (PP), determined by subtracting analyzed phytate phosphorus from analyzed total phosphorus.

and readjusted to the two test pHs of 2.5 or 6.5, prior to the start of the incubations. To a test tube with 3.0 mL of the substrate solution and added Ca, a 100-µL volume of phytase enzyme was added. The resulting mixtures were incubated at 37 °C for 0, 15, 30, 60, or 120 min. A 2-mL volume of ammonium molybdate-metavanadate reagent prepared according to Chen (15) was added to stop the reaction, and liberated P was measured spectrophotometrically at 410 nm (19), using inorganic P as a standard. Effect of Inorganic Micro-Mineral Premix. Five levels of IMM were used (0, 0.34, 1.7, 2.5, and 3.4 g/L). The levels used were chosen to reflect dietary additions of IMM at 0, 1.0, 5.0, 7.5, or 10.0 g/kg diet. These levels were selected based on the recommended inclusion level of this IMM in poultry diets of 1.0 g/kg and three levels higher (5.0, 7.5, and 10.0 g/kg), to exacerbate the potentially negative effect of micro-minerals. The solutions were incubated with the phytase enzyme and PP hydrolysis determined as described previously. Effect of Micro-Mineral-Amino Acid Complexes. The MAAC was added to the substrate at levels that supplied the same concentrations of Zn, Mn, Fe, Cu, I, and Co as those supplied by 0, 1.0, 5.0, 7.5, or 10.0 g/kg IMM in the diet (0, 4.5, 22.5, 33.75, and 45 g/kg MAAC). Phytate P hydrolysis in the presence of MAAC was determined as described previously for IMM. For the micro-mineral studies (IMM and MAAC), micro-mineral levels studied are identified as 0, 1, 5, 7.5, and 10X; where 1X is equal to 1 g/kg in the case of IMM and 4.5 g/kg in the case of MAAC. The 1X level of either IMM or MAAC supplied the same amount of micro-minerals (Fe, Mn, Zn, Cu, I, and Co). In Vivo Phytate Phosphorus Hydrolysis. Animals and Diets. A mash starter diet was mixed such that it met or exceeded National Research Council (20) broiler recommendations for all nutrients. For the experimental diets, a corn-soybean meal grower basal with no added micro-minerals, inorganic Ca, or P (Table 1) was mixed and analyzed for Ca, P, and PP, as described in the sample analysis section, before the experimental diets were formulated. The experimental design was a 2 × 3 factorial with two added Ca levels (0, 5.0 g/kg) and three micro-mineral sources (none, 1X IMM, and 1X MAAC; 1X of each as defined previously). The basal diet was used at 970 g/kg of the diet, and CaCO3, IMM, MAAC, and Celite were added to achieve levels desired in the experimental diets. Celite was added to all diets as an indigestible marker (21) and as a filler, to achieve 100% without affecting diet nutrient density, with a minimum inclusion rate of 10 g/kg. This level is the minimum needed for effective use of Celite as a marker. The dietary treatments were (1) basal, (2)

Phytate Phosphorus Hydrolysis basal plus 5 g/kg Ca (from CaCO3), (3) basal plus 1X IMM, (4) basal plus 1X IMM, plus 5.0 g/kg Ca (from CaCO3), (5) basal plus 1X MAAC, and (6) basal plus 1X MAAC plus 5.0 g/kg Ca (from CaCO3). Day-old male (Ross 308) broiler chicks were raised in floor pens from hatch to 20 days of age and fed the starter diet, ad libitum. On day 20, birds were randomly assigned to eight replicate battery pens per dietary treatment, four birds per pen. The six dietary treatments were assigned to battery pens, using a completely randomized design. Birds were fasted for 16 h and then fed the experimental diet ad libitum for 30 h, sacrificed by cervical dislocation, ileum separated, and ileal contents collected by expressing gently. The ileum was defined as the segment from the Meckel’s diverticulum to 3 cm before the ileocecal junction. Ileal contents were pooled by pen (the experimental unit), dried at 80 °C for 24 h, and stored at 10 °C for later analysis. The experimental period (30 h) was chosen to minimize functional adaptations of the gastrointestinal tract to the diets (22, 23). All guidelines of the Animal Care and Use Committee of the University of Maryland were followed. Sample Analyses. Diets and ileal samples were ground to pass through a 0.5 and 0.25 mm screen, respectively. On all samples, moisture (24), acid insoluble ash (25), Ca (26), P (19), and PP (according to the method of Rounds and Nielsen (27) as modified by Newkirk and Classen (28)) were determined. All analyses were done in duplicate. Statistical Analyses. For the in vitro experiment, the Ca effect was studied using a 4 × 2 factorial (four Ca levels, two pHs) design. The data were analyzed using mixed procedures of SAS (29). Pairwise comparisons were done to compare means within each time period/pH combination, using Tukey’s HSD test (30) to control experiment-wise error rate. For the micro-mineral study, the design was a 2 × 5 × 2 factorial (two pHs, five micro-mineral levels, two sources), and the data were analyzed separately for each time period, using mixed procedures of SAS (29). To test the effect of micro-mineral level, pairwise comparisons were done to compare means within each combination of pH, time, and source. The effect of micro-mineral source was also tested, by doing pairwise comparisons across sources within each time, pH, and level combination. Significance was accepted at P < 0.05. For the in vivo experiment, the design was a completely randomized 3 × 2 factorial (three micro-mineral sources and two Ca levels), resulting in six dietary treatments. Each pen served as an experimental unit (eight pens/treatment), and data were analyzed using the General Linear Models procedures of SAS (29). When the model was significant, treatment means were separated using Tukey’s HSD test (30). Significance was accepted at P < 0.05. RESULTS AND DISCUSSION

In Vitro Phytate Phosphorus Hydrolysis. The results of the in vitro studies are summarized in Table 2 and Figures 1, 2, and 3. In the Ca study, significant Ca, pH, and Ca × pH interaction was present at all time periods. Pairwise comparisons were done to test simple effect means, and results are summarized in Figure 1. At pH 2.5 (Figure 1A), PP hydrolysis was reduced (P < 0.05) by the addition of 4.0 g/kg or 9.0 g/kg Ca at all incubation times. A 1.0-g/kg Ca level resulted in significant reduction in PP hydrolysis only after 120 min of incubation. At pH 6.5, however, addition of Ca at levels as low as 1.0 g/kg resulted in a decrease (P < 0.05) in PP hydrolysis at 60 and 120 min, versus the no-Ca-added treatment (Figure 1B). Addition of Ca decreased PP hydrolysis to a greater extent at pH 6.5 than at 2.5, which explains the significant Ca × pH interaction. The addition of Ca at 1.0, 4.0, or 9.0 g/kg reduced PP hydrolysis during a 60 min incubation period by 2, 25, and 56%, respectively, at pH 2.5. At pH 6.5, the reductions were 23, 48, and 46%, respectively. These findings agree with published work, which showed that mineral cations, including Ca, negatively influenced PP hydrolysis and that this effect was more pronounced at higher pHs (6, 31, 32).

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Table 2. P Values from the ANOVA Analysis for the Effect of Micro-Mineral Level and Source on in Vitro Phytate Phosphorus Hydrolysis at pH 2.5 and 6.5 time (min) sources of variation levela sourceb pHc level × source level × pH source × pH level × source × pH

15

30

60

120